Fluorescent labels find use in variety of different biological, chemical, medical and biotechnological applications. One example of where such labels find use is in polynucleotide sequencing, particularly in automated DNA sequencing, which is becoming of critical importance to large scale DNA sequencing projects, such as the Human Genome Project.
In methods of automated DNA sequencing, differently sized fluorescently labeled DNA fragments which terminate at each base in the sequence are enzymatically produced using the DNA to be sequenced as a template. Each group of fragments corresponding to termination at one of the four labeled bases are labeled with the same label. Thus, those fragments terminating in A are labeled with a first label, while those terminating in G, C and T are labeled with second, third and fourth labels respectively. The labeled fragments are then separated by size in an electrophoretic medium and an electropherogram is generated, from which the DNA sequence is determined.
As method of automated DNA sequencing have become more advanced, of increasing interest is the use of sets of fluorescent labels in which all of the labels are excited at a common wavelength and yet emit one of four different detectable signals, one for each of the four different bases. Such labels provide for a number of advantages, including high fluorescence signals and the ability to electrophoretically separate all of the labeled fragments in a single lane of an electrophoretic medium which avoids problems associated with lane to lane mobility variation.
Although such sets of labels have been developed for use in automated DNA sequencing applications, heretofore the differently labeled members of such sets have each emitted at a different wavelength. Thus, conventional automated detection devices currently employed in methods in which all of the enzymatically produced fragments or primer extension products are separated in the same lane must be able to detect emitted fluorescent light at four different wavelengths. This requirement can prove to be an undesirable limitation. More specifically, carrying out sequencing on vast numbers of different DNA templates simultaneously increases the number of different fragments and corresponding labels required. At the same time, there is a need for a reduction in the complexity of the detection device, e.g. a device which can operate with light detection at only two wavelengths is preferable.
It would therefore be desirable to develop sets of fluorescent labels capable of providing four distinguishable signals, where the number of wavelengths associated with the four different signals is less than the number of different labels, e.g. where four different labels provide signals comprising emitted light at from one to two wavelengths. With such sets one could either: (1) reduce the complexity of automated detector devices or (2) increase the throughput of detectors capable of detecting at four different wavelengths, thereby achieving sequencing two DNA templates, or the same double stranded templates from both the 5' and 3' end, simultaneously.
Relevant Literature
DNA sequencing is reviewed in Griffin & Griffin, Appl. Biochem. Biotechnol. (1993) 38: 147-159. Fluorescence energy transfer labels and their use in DNA sequencing applications are described in Ju et al., Nucleic Acids Res. (1996) 24: 1144-1148, Ju et al., Nat. Med. (1996) 2: 246-249, Ju et al., Anal. Biochem. (1995) 231: 131-140. Ju et al., Proc. Natl. Acad. Sci. USA (1995) 92: 4347-4351. Use of fluorescent energy transfer labels for non-DNA sequencing multi component analysis application is described in Wang et al., Anal. Chem. (1995) 67: 1197-1203; Ziegle et al., Genomics (1992) 14: 1026-1031; and Repp et al., Leukemia (1995) 9: 210-215. Other references describing multi-component analysis applications include Schena et al., Science (1995) 270: 467-469.
Other references of interest include U.S. Pat. Nos. 4,996.143 and 5,326,692, as well as Glazer and Streyer, Biophys. J. (1983) 43: 383-386, Huang et al., Anal. Chem. (1992) 64: 2149-2154; Prober et al., Science ( 1987) 336-341; Smith et al., Nature (1986) 321: 674-679, Lu et al, J. Chromat. A (1994) 680: 497-501 and Ansorge et al., Nucleic Acids Res. (1987) 15: 4593-4603.